System and Method for Generating Three-Dimensional Images From Two-Dimensional Bioluminescence Images and Visualizing Tumor Shapes and Locations

ABSTRACT

A system and methods for generating 3D images from 2D bioluminescent images and visualizing tumor locations are provided. A plurality of 2D bioluminescent images of a subject are acquired using any suitable bioluminescent imaging system. The 2D images are registered to align each image and to compensate for differences between adjacent images. After registration, corresponding features are identified between consecutive sets of 2D images. For each corresponding feature identified in each set of 2D images, an orthographic projection model is applied, such that rays are projected through each point in the feature. The intersection points of the rays are plotted in a 3D image space. All of the 2D images are processed in the same manner, such that a resulting 3D image of a tumor is generated.

RELATED APPLICATIONS

This application is a continuation application of and claims the benefitof priority to U.S. patent application Ser. No. 12/065,980 filed Oct. 9,2008, now U.S. Pat. No. 8,218,836, which is a U.S. National PhaseApplication under 35 U.S.C. 371 of International Application No.PCT/US06/34320 filed Aug. 31, 2006, which claims the benefit of U.S.Provisional Application Ser. No. 60/715,610 filed Sep. 12, 2005, theentire disclosures of which are expressly incorporated herein byreference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to the generation of three-dimensionalimages, and more particularly, to a system and methods for generatingthree-dimensional images from two-dimensional bioluminescence images andvisualizing tumor shapes and locations.

2. Related Art

Bioluminescence imaging (BLI) is an emerging technique for sensitive andnon-invasive imaging, which can be used for monitoring molecular eventsin living animals. Important applications of this imaging techniqueinclude gene therapy and cell trafficking studies. Unlikefluorescence-based optical imaging approaches which require an externalsource of light for excitation of fluorophores, BLI generates atwo-dimensional (2D) view of gene expressions using a charge-coupleddevice (CCD) camera to detect the internal light produced by luciferases(catalysts in light generating reactions) through the oxidation of anenzyme-specific substrate (luciferin). The increasing use of BLI as apreferred modality for imaging small animals is due, in large part, tothe need for repeatedly imaging animals that have been transplanted withgene-marked cells. Other imaging modalities, such as positron emissiontomography (PET) and magnetic resonance imaging (MRI), are unsuitablefor repeated and rapid imaging in laboratory settings.

Recent research activities have focused on bioluminescence tomography(BLT) in an effort to extract depth information from 2D bioluminescenceimages. Such efforts, however, have been largely ineffective ingenerating useful 3D images with high resolution. The use of multipleCCD cameras to measure bioluminescence signals has been suggested, butthis approach is expensive and requires careful calibration of multiplecameras. As such, although BLI is a useful imaging modality forgenerating 2D images, there currently is no practical technique forrapidly generating three-dimensional (3D) images from a series of 2D BLIimages.

In addition to the aforementioned limitations, existing BLI techniquesdo not allow for the rapid and accurate visualization of the physicalshape and location of a tumor in a subject, in three dimensions. Whilethere has been extensive research on multi-modal image registration inthe image processing literature (based on matching geometric features orthe optimization of intensity-based energy functions), no practical, 3Dvisualization approach for BLI images has been developed. Moreover,images generated by existing BLI techniques cannot be easily registeredwith 3D images generated by other imaging modalities (such as computedtomography (CT) and microCT) so that the physical shape and location ofa tumor can be quickly discerned.

Accordingly, what would be desirable, but has not yet been provided, isa system and methods for generating three-dimensional images fromtwo-dimensional, bioluminescence images and visualizing tumor shapes andlocations.

SUMMARY OF THE INVENTION

The present invention relates to a system and methods for reconstructing3D images from 2D bioluminescent images and visualizing tumor shapes andlocations. A plurality of 2D bioluminescent images of a subject areacquired using any suitable bioluminescent imaging system. The 2D imagesare taken during a complete revolution about an axis of the subject,such that the imaging system is rotated about the subject by apredetermined angle between each image. Optionally, the imaging systemcould be held in a stationary position, the subject could be rotatedthrough a complete revolution, and a plurality of images could be taken.After imaging, the 2D images are registered according to the rotationaxis to align each image and to compensate for differences betweenadjacent images. After registration, corresponding features areidentified between consecutive sets of 2D images. For each correspondingfeature identified in each set of 2D images, an orthographic projectiontechnique (or model) is applied, such that rays are projected througheach point in the feature. The intersection points of the rays areplotted in a 3D image space. All of the 2D images are processed in thesame manner, such that the resulting 3D image of a tumor is generated.The subject itself, as well as a container holding the subject, can alsobe rendered in the 3D image using the orthographic projection technique.The tumor can be registered with a reference image of the subject or animage of the container so that the precise shape and location of thetumor can be visualized, together with detailed anatomical structureinformation extracted from other imaging modalities such as microCT.

BRIEF DESCRIPTION OF THE DRAWINGS

Other important objects and features of the present invention will beapparent from the following Detailed Description of the Invention, takenin connection with the accompanying drawings, in which:

FIG. 1 is a flowchart showing the method of the present invention forgenerating 3D images from 2D bioluminescence images and visualizingtumor shapes and locations;

FIGS. 2A-2C are photographs of 2D bioluminescence images of an animal;

FIG. 3A shows photographs of 2D bioluminescence images prior to imageregistration by the preset invention; FIG. 3B shows the photographs ofFIG. 3A after image registration by the present invention;

FIG. 4 is a flowchart showing processing step 22 of FIG. 1 in greaterdetail;

FIGS. 5A-5 b are photographs showing corresponding features identifiedby the present invention in two, sequential, 2D bioluminescent images;

FIG. 6 is a flowchart showing processing step 24 of FIG. 1 in greaterdetail;

FIGS. 7A-7B are diagrams illustrating the orthographic projection modelimplemented by the present invention for rendering a 3D image;

FIGS. 8A-8B are diagrams illustrating the orthographic projection modelof the present invention in greater detail;

FIG. 9A is a front view of a 3D tumor visualization generated by thepresent invention and superimposed on a 2D bioluminescence image; FIG.9B is a perspective view of the 3D tumor visualization of FIG. 9A, shownwith respect to the 2D bioluminescence image;

FIG. 10 shows photographs of microCT images of a mouse;

FIG. 11A is a front view of a 3D tumor visualization generated by thepresent invention and superimposed on a 2D bioluminescence image; FIG.11B is a perspective view of the 3D tumor visualization of FIG. 11A,shown with respect to the 2D bioluminescence image; FIG. 11C is a frontview showing the 3D tumor visualization of FIG. 11A superimposed on amicroCT image of a mouse;

FIGS. 12A-12C are photographs showing a segmentation technique appliedto 2D bioluminescence images for highlighting desired regions of theimages;

FIG. 13 is a diagram illustrating the orthographic projection techniqueof the present invention implemented using visual hulls;

FIGS. 14A-14B are sample images of 3D tumor visualizations generated bythe present invention; and

FIGS. 15A-15B are sample images of 3D tumor and specimen containervisualizations generated by the present invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a system and methods for generating (orreconstructing) 3D images from 2D bioluminescent images and visualizingtumor locations. A plurality of 2D bioluminescent images of a subjectare acquired during a complete revolution of an imaging system about asubject, using any suitable bioluminescent imaging system. Afterimaging, the 2D images are registered according to the rotation axis toalign each image and to compensate for differences between adjacentimages. After registration, corresponding features are identifiedbetween consecutive sets of 2D images. For each corresponding featureidentified in each set of 2D images, an orthographic projectiontechnique (or model) is applied, such that rays are projected througheach point in the feature. The intersection points of the rays areplotted in a 3D image space. All of the 2D images are processed in thesame manner, such that a resulting 3D image of a tumor is generated. The3D image can be registered with a reference image of a subject or acontainer holding the subject, so that the precise location of the tumorcan be visualized.

FIG. 1 is a flowchart showing the method of the present invention,indicated generally at 10, for visualizing 3D tumor locations from 2Dbioluminescence images. Beginning in step 12, a 2D bioluminescence imageof a subject is acquired. Preferably, the image is acquired using asuitable CCD-based bioluminescence imaging system, such as the IVISImaging System 100 Series or 200 Series manufactured by Xenogen, Inc.(Alameda, Calif.). The IVIS imaging system includes an ultra-low noiseCCD camera, a low background imaging chamber, a six-position opticalfilter wheel, and high-efficiency optics. It should be noted that anysuitable bioluminescence imaging system could be utilized. The subjectcould be a small animal or any other living organism.

In step 14, the imaging apparatus is rotated (i.e., moved along acircular path about an axis of the subject, such as the vertical axis)at a predetermined angle, while the subject is held in a stationaryposition. The angle of rotation could be adjusted as desired. It hasbeen found that relatively small angles of rotation (e.g., 12 degrees)have been found to be sufficient. The rotation angle is small betweenconsecutive image acquisitions so as to ensure the availability ofaccurate corresponding points in adjacent images, thereby facilitatingstereo-like reconstruction of a 3D image. This configuration is simpler,more flexible, and less expensive than using multiple cameras, since anydesired number of images can be acquired by adjusting the rotationangle. In step 16, an additional 2D bioluminescence image of the subjectis taken, at the new position. In step 18, a determination is made as towhether additional images are to be acquired. If a positivedetermination is made, steps 14-18 are repeated. Preferably, a pluralityof 2D bioluminescent images are taken over a complete revolution aboutan axis of the subject.

After all 2D images have been acquired, step 20 occurs, wherein the 2Dimages are registered according to the rotation axis. Due to noise andjittering of the image-capturing system during the rotation of thesubject, the set of 2D images acquired in steps 12-18 may not beperfectly aligned. To correct for these errors, an image-based methodfor registering the images is applied, such that projections of therotating axis on all images overlap in the image space. For thispurpose, an image dissimilarity objective function is defined based onmutual information, and the translation and rotation parameters for eachimage are recovered by minimizing the objective function. Suppose asource image is identified as f, and its adjacent target image is g. Inthe most general case, consider a sample domain Ω in the image domain ofthe source image f. The objective is to recover the parameters Θ=(Tx ,Ty , θ) of a global transformation A such that the mutual informationbetween fΩ=f (Ω) and g_(Ω) ^(A)=g(A(Θ;Ω)) is maximized. Here theparameters Tx and Ty are translation parameters in the x and ydirections respectively, and θ denotes the rotation angle. Thedefinition for such mutual information can be expressed by the followingequation:

MI(X ^(fΩ) , X ^(g) ^(Ω) ^(A) )=H[X ^(fΩ) ]+H[X ^(g) ^(Ω) ^(A) ]−H[X^(fΩ,g) ^(Ω) ^(A) ]  (1)

In the equation above, X denotes the intensity random variable and Hrepresents the differential entropy. The image dissimilarity objectivefunction can then be defined as:

E(A(Θ))=−MI(X ^(fΩ) , X ^(g) ^(Ω) ^(A) )  (2)

By minimizing the objective function E, the maximization of mutualinformation is achieved. The calculus of variations with a gradientdescent method is then used to minimize E and to recover thetransformation parameters Tx , Ty and θ.

After registration of all images in step 20, step 22 occurs, whereincorresponding features between consecutive 2D bioluminescence images arecomputed. Then, in step 24, a 3D image of a tumor (or other feature ofthe subject being studied) is constructed by applying an orthographicprojection model to the corresponding features of the 2D imagesidentified in step 22. The 3D image provides a detailed and accuraterepresentation of the tumor. The 3D image can be registered with anotherimage of the subject, such as a microCT image or a bioluminescence imageof the subject, so that the precise physical location of the tumor canbe visualized.

FIGS. 2A-2C are photographs of 2D bioluminescence images of a smallanimal (a mouse) having a tumor in the abdomen. The images were acquiredusing steps 12-18 of FIG. 1. The bioluminescence images were acquiredfollowing injection of D-luciferin (given i.p. at 150 mg/ml) and usingthe IVIS imaging system. Images were acquired in a standard mode with2×2 binning. In order to achieve specificity of the response in thez-axis, an experimental configuration was tested. The animal to beimaged was inserted into a cylindrical 50 ml tube cut at both ends,which can be rotated by a small angle (12 degrees) about the verticalaxis of the tube. Images were acquired at every rotation stage clockwisefrom the vertical axis. This generated a series of images including theoriginal image prior to any rotation. For small animals such as mice, a50 ml tube cut at both ends and at the bottom can be used as a holder.The anesthetized animal fits easily in the tube and can be placed in theimaging device without any discomfort. The animal can be rotated and 36images can be acquired. An added advantage of the 50 ml tube is that itcan be fitted with a soft foam to make the animal fit snugly into thetube, and the outside of the tube can be marked with fiduciary markersfor anatomical reference.

The images shown in FIGS. 2A-2C were generated after each small angle(e.g., 12 degrees) rotation. The intensity representation denotes thelevel of response in different locations. The bright reflections (due tothe tube surface) were eliminated using a pre-processing filtration stepbefore applying the method of the present invention. It should be notedthat the camera could also be held stationary, and the subject rotatedas necessary. The tumor regions have higher intensity values in the BLIimages. The dimensions along the long axis and short axis of the mousetumor were 1:2 cm, 1:1 cm and 1:1 cm. The imaging period lastedapproximately 20 minutes after injection of D-luciferin.

FIG. 3A shows photographs of 2D bioluminescence images prior to imageregistration performed by step 20 of FIG. 1. These images were generatedin a phantom study, wherein lysates from cells transduced with amammalian expression vector containing luciferase were embedded inagarose plugs and incubated with the substrate D-luciferin inside a tubecontaining 1% agarose in Tris Acetate EDTA buffer of pH 7.5. The imagesshown in FIG. 3A were generated after each successive rotation of thecamera of approximately 11.25 degrees, and are slightly skewed. Afterregistration by the present invention, the images are properly aligned,as shown in FIG. 3B.

FIG. 4 is a flowchart showing processing step 22 of FIG. 1 in greaterdetail. In step 22, corresponding features between successive images areidentified. In step 30, corner features on an image I are detected. Thisis achieved using the spatial image gradient (i.e. first orderderivatives), [Ix , Iy]. For a neighborhood Q surrounding a pixel p, amatrix C is formed, defined as:

$\begin{matrix}{C = \begin{pmatrix}{\sum I_{x}^{2}} & {\sum{I_{x}I_{y}}} \\{\sum{I_{x}I_{y}}} & {\sum I_{y}^{2}}\end{pmatrix}} & (3)\end{matrix}$

where the sums are taken over the neighborhood Q. Then, principalcomponent analysis is applied to compute the two eigenvalues λ1 and λ2(λ1≧λ2) of the matrix C, and corner features are identified as thoseneighborhoods where λ1≧λ2>0 and the smaller eigenvalue λ2 is larger thana threshold. Then, in step 32, regions around the corner features aredefined, and in step 34, a multi-modal similarity measure is applied tocalculate similarities in the regions. Such a measure is utilizedbecause nonlinear changes exist in feature appearances due to planarprojection after rotation. In step 36, a determination is made as towhether the measured similarity is above a threshold. If a positivedetermination is made, the regions are identified in step 38 as beingsimilar. Then, in step 40, a determination is made as to whether allcorner features have been processed. If a negative determination ismade, step 30 is re-invoked, so that additional corner features can beprocessed.

FIGS. 5A-5 b are photographs showing corresponding features identifiedby the present invention in two, sequential, 2D bioluminescent images.The numbers in the images identify corresponding features that have beencalculated by the present invention. The image shown in FIG. 5B wastaken at a rotation angle of 11.5 degrees from the image shown in FIG.5A.

FIG. 6 is a flowchart showing processing step 24 of FIG. 1 in greaterdetail. In step 24, a 3D image of a tumor is constructed by applying anorthographic projection model to the corresponding features identifiedin step 22 of FIG. 1. In step 42, the current 2D image and a subsequent2D image are loaded. Then, in step 44, a feature point common to bothimages (e.g., a pixel within a corresponding feature common to bothimages and identified in step 22) is identified. Then, in step 46, raysare projected through the feature points of both images. In step 48, thepoint of intersection of both rays is plotted in a 3D image space. Theray projection technique can be implemented in any suitable digitalcomputer system, using known techniques that are similar to ray tracingtechniques implemented in computer graphics.

In step 50, a determination is made as to whether additional featurepoints exist. If a positive determination is made, steps 44-50 arerepeated. If a negative determination is made, step 52 is invoked,wherein a determination is made as to whether additional images are tobe processed. If a positive determination is made, steps 42-50 arere-invoked. In this manner, all feature points for all images areplotted in the 3D image space. The resulting 3D image is ahigh-resolution visualization of one or more tumors in the subject. Thisrendering approach is equivalent to having multiple cameras surroundinga static object. However, it is much simpler and does not require thecalibration of multiple cameras.

FIGS. 7A-7B are diagrams illustrating the orthographic projection modelimplemented by the method of the present invention for rendering a 3Dimage. As shown in FIG. 7A, a series of 2D bioluminescent images 60 thathave been processed in accordance with the present invention (i.e., toregister the images and to identify corresponding features in adjacentimages) are used to plot one or more features 68 in a common volume 62of a 3D image space. For points in each common feature, such as points64 a and 64 b, rays, such as rays 66 a and 66 b are projected throughthe points. The intersection of the rays 66 a and 66 b is plotted in thecommon volume 62. As shown in FIG. 7B, this process is repeated for eachpoint of each common feature of each 2D image 70, about the entirecircumference of the common volume 72. The resulting 3D image in thecommon volume 72 represents a visualization of the tumor in the subject.It should be noted that this technique could be implemented to visualizeany desired feature of the subject.

FIGS. 8A-8B are diagrams illustrating the orthographic projection modelof the present invention in greater detail. In FIG. 8A, two consecutive2D images 80 a and 80 b are shown. Points 82 a and 82 b in the imagescorrespond to locations on the tube containing the subject. Points 86 aand 86 b correspond to locations of the tube center. To determine the 3Dlocation of these points, rays 83 a and 83 b are projected throughpoints 82 a and 82 b, respectively. The intersection point 84 of therays 83 a and 83 b corresponds to the 3D location of the points 82 a and82 b of the images 80 a and 80 b. Similarly, rays 85 a and 85 areprojected through the points 86 a and 86 b, respectively. Theintersection point 88 corresponds to the 3D location of the points 86 aand 86. Using this technique, a 3D image can be generated for anydesired feature in the 2D images 80 a and 80 b. Thus, for example, notonly can a tumor be imaged in 3 dimensions, but also the subject itself,any desired feature of the subject, or the container holding thesubject. Advantageously, this allows for precise, 3D visualization oftumor locations with respect to the subject's anatomy.

FIG. 8B shows the orthographic projection technique of the presentinvention applied to generate a 3D image corresponding to the center ofa tumor. Rays 94 a and 94 b are projected through tumor center locations92 a and 92 b in the 2D images 90 a and 90 b, respectively. Theintersection point 96 is mapped in a 3D image space, and corresponds tothe location of the center of the tumor. The tumor center locations 92 aand 92 b in the 2D images 90 a and 90 b can be computed as the centroidof high-intensity signal regions in the images 90 a and 90 b.

In the aforementioned phantom study, since the object surface can beapproximated using a cylinder, it is possible to determine the radius ofthe cylinder using the recovered 3D points on the tube surface, and torender the surface as a cylinder. Then, a relationship is establishedbetween the reconstructed object dimension measurements in theobject-centered reference frame and that in the physical world. This isachieved by computing the conversion ratio based on one basemeasurement, such as the diameter or the length of the tube (or thesubject). Such an approximation is shown in FIG. 9A, which is a frontview of a 3D visualization of a tumor 102 generated by the presentinvention and superimposed on a 2D bioluminescence image 100 of thesubject 104. As shown in FIG. 9B, the visualization also includes a 3Drendering of a cylinder 106, which corresponds to the tube holding thesubject.

In the phantom study mentioned above, validation of the 3D visualizationgenerated by the present invention was achieved by comparing thevisualized 3D location of the tumor center with images of the subjecttaken with a microCT scanner. For purposes of validation, the 3Dvisualization shown in FIGS. 9A-9B was compared to microCT images of thesame subject. The 3D visualization generated by the present inventionindicates that the tumor center location is within the tube containingthe specimen. The microCT images confirmed that this visualization isaccurate. Further, physical measurements indicated that the 3D distancebetween the true luciferase-positive cell lysates center and thevisualized 3D center are within 2 mm of each other, thus indicating ahigh degree of accuracy.

The present invention was also tested on a mouse to determine itsfeasibility for use in small animal tests, as well as to demonstrate theability of the present invention to register the 3D visualization with amicroCT image of an animal. The results of this test are illustrated inFIGS. 10 and 11A-11C. In preparation for the test, the mouse wasanesthetized with isoflurane inhalation, injected with 150 mg/kg ofD-luciferin, and immobilized in an open 50 ml tube and placed on theimaging stage of the IVIS imaging system. The mouse was then imaged, andexamples of the resulting BLI images are shown in FIG. 2. The mouse wasthen carried over to the microCT machine, in the same position whileremaining under isoflurane anesthesia. FIG. 10 shows microCT images ofthe mouse. The present invention was then applied to the BLI images,resulting in the 3D visualizations shown in FIGS. 11A-11B. In FIG. 11A,a front view of the visualized tumor 112 is shown superimposed on asingle BLI image 110 of the mouse. In FIG. 11B, the visualized tumor 112is shown in perspective view, within the visualized cylinder 116corresponding to the tube holding the mouse. A plurality of 2D BLIimages 114 are shown for reference. Then, as shown in FIG. 11C, thevisualized tumor 112 was superimposed on a single microCT image 120 ofthe mouse. The visualized tumor 112 was thus registered with the microCTimage, resulting in a highly accurate indication of the physicallocation of the tumor inside the mouse's body. Registration with themicroCT image can be achieved using landmark information and anInterative Closest Point (ICP) technique, or any other suitabletechnique. Repeated imaging of the a subject over time can also beperformed, so that temporal information can be visualized (e.g., to showchanges in anatomical structures over time).

FIGS. 12A-12C are photographs showing a segmentation technique appliedto 2D bioluminescence images for highlighting desired regions of theimages. Segmenting the image to highlight desired portions has beenfound to particularly useful where the orthographic projection techniqueof the present invention is implemented using visual hulls, since thevisual hull technique depends both on object silhouettes and on thecamera viewing direction. FIG. 12A shows the original 2D BLI images. Inorder to facilitate correct segmentation, a monochromatic background wascaptured to distinguish the tube containing the small animal from theenvironment in the experiment setup. First, the contour (or silhouette)of the tube containing the small animal was extracted from the inputimages by simple thresholding. FIG. 12B shows the tube segmentationresult that were obtained. Then, according to the characteristic oftumor in the BLI images (which appear as higher intensities), the tumorwas then segmented from the tube region by combining tumor intensity andedge information. FIG. 12C shows segmentation results of the tumor inthe images.

FIG. 13 is a diagram illustrating the orthographic projection techniqueof the present invention implemented using visual hulls. Formallydefined, the visual hull of an object S with respect to the viewingregion R, denoted by V H(S;R), is a volume in space such that for eachpoint P in V H(S;R) and each viewpoint V in R, the half-line from Vthrough P contains at least one point of S. This definition states thatthe visual hull consists of all points in space whose images lie withinall silhouettes viewed from the viewing region. Stated another way, thevisual hull is the maximal object that has the same silhouettes as theoriginal object, as viewed from the viewing region. In the presentinvention, the segmented object and tumor silhouettes are projected intothe 3D space by cylindrical visual hulls, illustratively indicated byreference numeral 130 in FIG. 13. By computing the intersection of thevisual hulls projected from all images (i.e., all viewing directions),an estimation of the shape and location of the animal and its interiortumors can be obtained.

FIGS. 14A-14B are sample images of 3D tumor visualizations generated bythe present invention. As can be appreciated, tumors of various shapesand sizes can be visualized.

FIGS. 15A-15B are sample images of 3D tumor and specimen containervisualizations generated by the present invention. By visualizing boththe tumor and the entire container, the position of the tumor withrespect to the longitudinal axis of the container can be visualized, aswell as the approximate distance between the tumor and the containerwall.

To implement the invention, the following hardware could be used:bioluminescence imaging hardware, which could include anybioluminescence imaging system with upgrades to allow for rotation ofthe subject being imaged or rotation of the camera of the imaging systemabout the subject; a microCT scanner to acquire microCT images of thesubject; and suitable computer hardware, such as high-end computerworkstations, for data processing, rendering, and visualization.Computer software that could be utilized to implement the methods of thepresent invention includes, but is not limited to, Matlab, MicrosoftVisual Studio, and computer graphics libraries. Such software could beutilized to process the BLI, microCT, and ground truth annotation data,as well as to visualize the co-registration and reconstructed tumorvolume results.

It is conceivable that software coded to carry out the method of thepresent invention could be integrated for use with anycommercially-available bioluminescent imaging system (or other imagingsystem), to provide a complete, 3D visualization system. Moreover, themethod of the present invention can be extended to register the centerof mass of several areas near the maximum response regions of images,where the intensity is 5-10% lower from this maximum, so as to providean estimate of the tumor enclosing volume. The present invention hasmultiple applications, such as the study of cell trafficking, tumorgrowth, in vivo patient response to therapy, studies relating tohematological reconstitution following bone marrow transplantation, andother applications.

Having thus described the invention in detail, it is to be understoodthat the foregoing description is not intended to limit the spirit andscope thereof. What is desired to be protected by Letters Patent is setforth in the following claims.

1. A method for generating a three dimensional image from a plurality oftwo-dimensional bioluminescence images, comprising: acquiring aplurality of two-dimensional bioluminescence images using an imagingsystem; processing the plurality of two-dimensional bioluminescenceimages so that adjacent images are aligned with each other; identifyingat least one feature common to each of the plurality of two-dimensionalbioluminescence images; and applying an orthographic projection model tothe at least one feature of each of the plurality of two-dimensionalbioluminescence images to generate a three-dimensional image of the atleast one feature.
 2. The method of claim 1, wherein the step ofacquiring the plurality of two-dimensional bioluminescence imagescomprises taking a first two-dimensional bioluminescence image of asubject at a first location using an imaging system.
 3. (canceled) 4.(canceled)
 5. The method of claim 2, further comprising rotating asubject at a predetermined angle and taking a second two-dimensionalimage of a subject.
 6. (canceled)
 7. (canceled)
 8. (canceled)
 9. Themethod of claim 1, wherein the step of applying the orthographicprojection model to the at least one feature comprises identifyingfeature points in the plurality of two-dimensional bioluminescenceimages corresponding to the at least one feature.
 10. The method ofclaim 9, further comprising projecting rays through the feature points.11. The method of claim 10, further comprising plotting an intersectionpoint of the rays in the three-dimensional image.
 12. The method ofclaim 9, further comprising projecting visual hulls through the featurepoints.
 13. The method of claim 12, further comprising plotting anintersection point of the visual hulls in the three-dimensional image.14. (canceled)
 15. The method of claim 1, further comprising registeringthe three-dimensional image with a reference image to visualize alocation of the at least one feature.
 16. A system for generating athree-dimensional image from a plurality of two-dimensionalbioluminescence images, comprising: an imaging system for acquiring aplurality of two-dimensional bioluminescence images; means forprocessing the plurality of two-dimensional bioluminescence images sothat adjacent images are aligned with each other; means for identifyingat least one feature common to each of the plurality of two-dimensionalbioluminescence images; and an orthographic projection model applied tothe at least one feature of each of the plurality of two-dimensionalbioluminescence images to generate a three-dimensional image of the atleast one feature.
 17. The system of claim 16, wherein the imagingsystem acquires a first two-dimensional bioluminescence image of asubject at a first location.
 18. (canceled)
 19. (canceled)
 20. Thesystem of claim 17, wherein the imaging system rotates a subject at apredetermined angle and acquires a second two-dimensionalbioluminescence image of a subject.
 21. (canceled)
 22. (canceled) 23.(canceled)
 24. The system of claim 16, wherein the orthographicprojection model identifies feature points in the plurality oftwo-dimensional bioluminescence images corresponding to the at least onefeature.
 25. The system of claim 24, wherein the orthographic projectionmodel projects rays through the feature points.
 26. The system of claim25, wherein the orthographic projection model plots an intersectionpoint of the rays in the three-dimensional image.
 27. The system ofclaim 24, wherein the orthographic projection model projects visualhulls through the feature points.
 28. The system of claim 27, whereinthe orthographic projection model plots an intersection point of thevisual hulls in the three-dimensional image.
 29. (canceled)
 30. Thesystem of claim 16, further comprising means for registering the threedimensional image with a reference image to visualize a location of theat least one feature.
 31. A method for visualizing a three-dimensionaltumor location, comprising: acquiring a plurality of two-dimensionalbioluminescence images of a tumor using an imaging system; processingthe plurality of two-dimensional bioluminescence images so that adjacentimages are aligned with each other; identifying at least one featurecommon to each of the plurality of two-dimensional bioluminescenceimages; applying an orthographic projection model to the at least onefeature of each of the plurality of two-dimensional bioluminescenceimages to generate a three-dimensional image of the tumor; andregistering the three-dimensional image with a reference image tovisualize a location of the tumor.
 32. The method of claim 31, whereinthe step of acquiring the plurality of two-dimensional bioluminescenceimages comprises taking a first two-dimensional bioluminescence image ofa subject at a first location using an imaging system.
 33. (canceled)34. (canceled)
 35. The method of claim 32, further comprising rotating asubject at a predetermined angle and taking a second two-dimensionalbioluminescence image of a subject.
 36. (canceled)
 37. (canceled) 38.(canceled)
 39. The method of claim 31, wherein the step of applying theorthographic projection model to the at least one feature comprisesidentifying feature points in the plurality of two-dimensionalbioluminescence images corresponding to the at least one feature. 40.The method of claim 39, further comprising projecting rays through thefeature points.
 41. The method of claim 40, further comprising plottingan intersection point of the rays in the three-dimensional image. 42.The method of claim 39, further comprising projecting visual hullsthrough the feature points.
 43. The method of claim 42, furthercomprising plotting an intersection point of the visual hulls in thethree-dimensional image.
 44. The method of claim 31, further comprisingsegmenting specific features of the plurality of two-dimensionalbioluminescence images.
 45. A system for visualizing a three-dimensionaltumor location, comprising: an imaging system for acquiring a pluralityof two-dimensional bioluminescence images of a tumor; means forprocessing the plurality of two-dimensional bioluminescence images sothat adjacent images are aligned with each other; means for identifyingat least one feature common to each of the plurality of two-dimensionalbioluminescence images; an orthographic projection model applied to theat least one feature of each of the plurality of two-dimensionalbioluminescence images to generate a three-dimensional image of thetumor; and means for registering the three-dimensional image with areference image to visualize a location of the tumor.
 46. The system ofclaim 45, wherein the imaging system acquires a first two-dimensionalbioluminescence image of a subject at a first location.
 47. (canceled)48. (canceled)
 49. The system of claim 45, wherein the imaging systemrotates a subject at a predetermined angle and acquires a secondtwo-dimensional bioluminescence image of a subject.
 50. (canceled) 51.(canceled)
 52. (canceled)
 53. The system of claim 45, wherein theorthographic projection model identifies feature points in the pluralityof two-dimensional bioluminescence images corresponding to the at leastone feature.
 54. The system of claim 53, wherein the orthographicprojection model projects rays through the feature points.
 55. Thesystem of claim 54, wherein the orthographic projection model plots anintersection point of the rays in the three-dimensional image.
 56. Thesystem of claim 53, wherein the orthographic projection model projectsvisual hulls through the feature points.
 57. The system of claim 56,wherein the orthographic projection model plots an intersection point ofthe visual hulls in the three-dimensional image.
 58. The system of claim55, further comprising means for segmenting specific features of theplurality of two-dimensional bioluminescence images.